Metabolism of 3-chloro-4-fluoroaniline in rat using [14C]-radiolabelling, 19F-NMR spectroscopy, HPLC-MS/MS, HPLC-ICPMS and HPLC-NMR

The metabolic fate of 3-chloro-4-fluoroaniline was investigated in rat following intraperitoneal (i.p.) administration at 5 and 50 mg kg(-1) using a combination of HPLC-MS, HPLC-MS/MS, (19)F-NMR spectroscopy, HPLC-NMR spectroscopy and high-pressure liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) with (35)Cl and (34)S detection. The metabolism of 3-chloro-4-fluoroaniline at both doses was rapid and extensive, to a large number of metabolites, with little unchanged compound excreted via the urine. Dosing at 5 mg kg(-1) with [(14)C]-labelled compound enabled the comparison of standard radioassay analysis methods with (19)F-NMR spectroscopy. (19)F-NMR resonances were only readily detectable in the 0-12 h post-dose samples. Dosing at 50 mg kg(-1) allowed the facile and specific detection and quantification of metabolites by (19)F-NMR spectroscopy. Metabolite profiling was also possible at this dose level using HPLC-ICPMS with (35)Cl-specific detection. The principal metabolites of 3-chloro-4-fluoroaniline were identified as 2-amino-4-chloro-5-fluorophenyl sulfate and 2-acetamido-4-chloro-5-fluorophenyl glucuronide. N-acetylation and hydroxylation followed by O-sulfation were the major metabolic transformations observed.     

Metabolism of 3-chloro-4-fluoroaniline in rat using [14C]-radiolabelling, 19F-NMR spectroscopy,HPLC-MS/MS,HPLC-ICPMS and HPLC-NMR

The metabolic fate of 3-chloro-4-fluoroaniline was investigated in rat following intraperitoneal (i.p.) administration at 5 and 50?mg?kg^?1 using a combination of HPLC-MS, HPLC-MS/MS, ¹⁹F-NMR spectroscopy, HPLC-NMR spectroscopy and high-pressure liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) with ³⁵Cl and ³⁴S detection. The metabolism of 3-chloro-4-fluoroaniline at both doses was rapid and extensive, to a large number of metabolites, with little unchanged compound excreted via the urine. Dosing at 5?mg?kg^?1 with [¹⁴C]-labelled compound enabled the comparison of standard radioassay analysis methods with ¹⁹F-NMR spectroscopy. ¹⁹F-NMR resonances were only readily detectable in the 0–12?h post-dose samples. Dosing at 50?mg?kg^?1 allowed the facile and specific detection and quantification of metabolites by ¹⁹F-NMR spectroscopy. Metabolite profiling was also possible at this dose level using HPLC-ICPMS with ³⁵Cl-specific detection. The principal metabolites of 3-chloro-4-fluoroaniline were identified as 2-amino-4-chloro-5-fluorophenyl sulfate and 2-acetamido-4-chloro-5-fluorophenyl glucuronide. N-acetylation and hydroxylation followed by O-sulfation were the major metabolic transformations observed.     

19F and 1H magnetic resonance strategies for metabolic studies on fluorinated xenobiotics: application to flurbiprofen [2-(2-fluoro-4-biphenylyl)propionic acid

Strategies for the use of 1H and 19F nuclear magnetic resonance (NMR) spectroscopy as an aid to the study of the metabolic fate of fluorinated drugs are discussed with reference to the application of these methods to flurbiprofen metabolism in man. 1H and 19F NMR analysis of untreated urine enabled the detection of two major and eight minor metabolites of the drug. The two major metabolites were identified using a combination of NMR spectroscopy, solid-phase extraction chromatography with 19F and 1H NMR detection and chemical hydrolysis to a flurbiprofen glucuronide and the glucuronide of the 4-hydroxy metabolite. 1H-19F 2D shift correlated spectroscopy and spin-echo difference experiments are discussed in relation to their use in the structural identification of drug metabolites.     

Metabolism of 2-fluoro-4-iodoaniline in earthworm Eisenia veneta using (19)F-NMR spectroscopy, HPLC-MS, and HPLC-ICPMS ((127)I)

A combination of (19)F-NMR spectroscopy, HPLC-MS/MS, HPLC-MS with constant neutral loss scanning of 127, and HPLC-ICPMS with iodine detection has enabled the profiling, quantification, and limited characterization of the metabolites produced in the earthworm Eisenia veneta, following exposure to 2-fluoro-4-iodoaniline. Mass spectrometric analysis of the worm tissue and coelomic fluid afforded the identification of two Phase II metabolites, N-glutamyl and N-glucoside conjugates, indicating the importance of these pathways in the detoxification of xenobiotics for earthworms. Several further metabolites were observed and quantified by (19)F-NMR spectroscopy and HPLC-(127)I-ICPMS, although these were of low abundance and their structures were not unequivocally identified. The parent compound and the glutamyl conjugate were found to be the major xenobiotic components of both the coelomic fluid and the worm tissue, representing approximately 23 and approximately 35%, respectively, of the dose that was recovered from the earthworm tissue extract.     

UPLC-MS,HPLC-radiometric,and NMR-spectroscopic studies on the metabolic fate of 3-fluoro-[U-14C]-aniline in the bile-cannulated rat

1. A study of the rates and routes of excretion of 3-fluoro-[U-¹⁴C]aniline following intraperitoneal administration to male bile-cannulated rats by liquid scintillation counting (LSC) gave a total recovery of ～ 90% in the 48?h following dosing, with the majority of the dose being excreted in the urine during the first 24?h (～ 49%).

2. The total recovery as determined by ¹⁹F-nuclear magnetic resonance (¹⁹F-NMR) was ～ 49%, with the majority of the dose excreted in the first 24?h (～ 41%). The comparatively low recovery in comparison to that obtained from LSC was due to matrix effects in bile and a contribution from metabolic defluorination.

3. High-performance liquid chromatography with radiometric profiling of urine and bile revealed a complex pattern of metabolites with the bulk of the dose excreted as a single peak.

4. Ultra-performance liquid chromatography-orthogonal acceleration time of flight mass spectrometry profiling also showed a complex pattern of metabolites, detecting ～ 21 metabolites of 3-fluoroaniline (3-FA) with six of these detected only in urine and four solely in bile.

5. ¹⁹F-NMR revealed the presence of the parent compound and 15 metabolites in urine collected during the first 24?h after -dosing. The matrix effects of bile on ¹⁹F-NMR spectroscopy made metabolite profiling impractical for this biofluid.

6. The major metabolite of 3-FA was identified as 2-fluoro-4-acetamidophenol-sulfate.

     

Application of (1)H- and (19)F-NMR spectroscopy in the investigation of the urinary and biliary excretion of 3,5-, 2,4-ditrifluoromethylbenzoic and pentafluorobenzoic acids in rat

1. The metabolism and excretion of 2,4-, 3,5-ditrifluoromethyl- and pentafluorobenzoic acids were studied in the bile-cannulated rat using (1)H- and (19)F-NMR spectroscopy following intraperitoneal administration at 50 mg kg(-1). 2. Pentafluorobenzoic acid was excreted in the urine entirely unchanged. No detectable compound or metabolites were eliminated in the bile. A total of 63.5 +/- 6.7% of the dose was recovered in the 24-h collection period. 3. In the case of 2,4-ditrifluromethyl benzoic acid, 83.9 +/- 5.2% of the dose was recovered in the 24h after administration, with about 52% being excreted in the urine and 32% in the bile. The majority of the material present in the urine was unchanged parent compound. In bile, some 60% of the compound-related material excreted was present as transacylated ester glucuronide conjugates. 4. For 3,5-ditrifluoromethylbenzoic acid, 49.6 +/- 5.3% of the dose was recovered in the 24-h collection period, with about 22% being excreted in the urine and 28% in the bile. The material excreted in both the urine and bile was a mixture of the parent acid and transacylated ester glucuronides. 5. Urinary excretion in bile-cannulated animals was similar to that found in studies using non-cannulated animals dosed at 100mg kg(-1).     

Metabolism of 2-fluoro-4-iodoaniline in earthworm Eisenia veneta using 19F-NMR spectroscopy,HPLC-MS,and HPLC-ICPMS (127I)

A combination of ¹⁹F-NMR spectroscopy, HPLC-MS/MS, HPLC-MS with constant neutral loss scanning of 127, and HPLC-ICPMS with iodine detection has enabled the profiling, quantification, and limited characterization of the metabolites produced in the earthworm Eisenia veneta, following exposure to 2-fluoro-4-iodoaniline. Mass spectrometric analysis of the worm tissue and coelomic fluid afforded the identification of two Phase II metabolites, N-glutamyl and N-glucoside conjugates, indicating the importance of these pathways in the detoxification of xenobiotics for earthworms. Several further metabolites were observed and quantified by ¹⁹F-NMR spectroscopy and HPLC-¹²⁷I-ICPMS, although these were of low abundance and their structures were not unequivocally identified. The parent compound and the glutamyl conjugate were found to be the major xenobiotic components of both the coelomic fluid and the worm tissue, representing approximately 23 and approximately 35%, respectively, of the dose that was recovered from the earthworm tissue extract.     

19 F-NMR and directly coupled 19 F/ 1 H-HPLC-NMR spectroscopic investigations of the metabolism of the model ecotoxin 3-trifluoromethylaniline in the earthworm species Eisenia veneta

1. The metabolic fate of the model ecotoxin 3-trifluoromethylaniline (3-TFMA) in earthworm was studied by 19 F- and directly coupled 19 F/ 1 H-HPLC-NMR spectroscopy. Earthworms of Eisenia veneta spp. were subjected to the ecotoxin during a filter papercontact toxicity test at exposure levels of 1000, 100, 10, 1 and 0.1 µg cm -2. A metabolic profile was obtained previously by 19 F-NMR spectroscopy and metabolites were observed at all the exposure levels. 2. Identification of metabolites in individual worm extracts at the (lethal) exposure levels of 1000 and 100 µg cm -2 could be achieved on-line without sample preparation by 19 F/ 1 H-HPLC-NMR spectroscopy. 19 F-HPLC-NMR spectroscopy was used in the continuous-flow mode, which enabled the HPLC chromatographic retention times (t R) of the metabolites to be established in a single analytical step. 3. In total, three 19 F-NMR signals could be detected, of which one was identified as the parent compound. Two earlier eluting metabolites were identified to be α - and β -glucoside conjugates of 3-TFMA. 4. Metabolites at the lower (sublethal) exposure levels of 10, 1 and 0.1 µg cm -2 escaped identification by 19 F/ 1 H-HPLC-NMR spectroscopy as outlined here and will require concentration prior to analysis.     

19F-NMR and directly coupled 19F/1H-HPLC-NMR spectroscopic investigations of the metabolism of the model ecotoxin 3-trifluoromethylaniline in the earthworm species Eisenia veneta

1. The metabolic fate of the model ecotoxin 3-trifluoromethylaniline (3-TFMA) in earthworm was studied by (19)F- and directly coupled (19)F/(1)H-HPLC-NMR spectroscopy. Earthworms of Eisenia veneta spp. were subjected to the ecotoxin during a filter papercontact toxicity test at exposure levels of 1000, 100, 10, 1 and 0.1 micro g cm(-2). A metabolic profile was obtained previously by (19)F-NMR spectroscopy and metabolites were observed at all the exposure levels. 2. Identification of metabolites in individual worm extracts at the (lethal) exposure levels of 1000 and 100 micro g cm(-2) could be achieved on-line without sample preparation by (19)F/(1)H-HPLC-NMR spectroscopy. (19)F-HPLC-NMR spectroscopy was used in the continuous-flow mode, which enabled the HPLC chromatographic retention times (t(R)) of the metabolites to be established in a single analytical step. 3. In total, three (19)F-NMR signals could be detected, of which one was identified as the parent compound. Two earlier eluting metabolites were identified to be alpha- and beta-glucoside conjugates of 3-TFMA. 4. Metabolites at the lower (sublethal) exposure levels of 10, 1 and 0.1 micro g cm(-2) escaped identification by (19)F/(1)H-HPLC-NMR spectroscopy as outlined here and will require concentration prior to analysis.     

Application of 1 H- and 19 F-NMR spectroscopy in the investigation of the urinary and biliary excretion of 3,5-, 2,4-ditrifluoromethylbenzoic and pentafluorobenzoic acids in rat

1. The metabolism and excretion of 2,4-, 3,5-ditrifluoromethyl- and pentafluorobenzoic acids were studied in the bile-cannulated rat using 1 H- and 19 F-NMR spectroscopy following intraperitoneal administration at 50?mg kg -1. 2. Pentafluorobenzoic acid was excreted in the urine entirely unchanged. No detectable compound or metabolites were eliminated in the bile. A total of 63.5 ± 6.7% of the dose was recovered in the 24-h collection period. 3. In the case of 2,4-ditrifluromethyl benzoic acid, 83.9 ± 5.2% of the dose was recovered in the 24h after administration, with about 52% being excreted in the urine and 32% in the bile. The majority of the material present in the urine was unchanged parent compound. In bile, some 60% of the compound-related material excreted was present as transacylated ester glucuronide conjugates. 4. For 3,5-ditrifluoromethylbenzoic acid, 49.6 ± 5.3% of the dose was recovered in the 24-h collection period, with about 22% being excreted in the urine and 28% in the bile. The material excreted in both the urine and bile was a mixture of the parent acid and transacylated ester glucuronides. 5. Urinary excretion in bile-cannulated animals was similar to that found in studies using non-cannulated animals dosed at 100mg kg -1.     

Pharmacokinetics,metabolism and excretion of the glycine antagonist GV150526A in rat and dog

1. The pharmacokinetics, metabolic fate and excretion of 3-[-2(phenylcarbamoyl) ethenyl-4,6-dichloroindole-2-carboxylic acid (GV150526), a novel glycine antagonist for stroke, in rat and dog following intravenous administration of [C14]-GV150526A were investigated. 2. Studies were also performed in bile duct-cannulated animals to confirm the route of elimination and to obtain more information on metabolite identity. 3. Metabolites in plasma, urine and bile were identified by HPLC-MS/MS and NMR spectroscopy. 4. GV150526A was predominantly excreted in the faeces via the bile, with only trace metabolites of radioactivity in urine (< 5%). Radioactivity in rat bile was predominantly due to metabolites, whereas approximately 50% of the radioactivity in dog bile was due to parent GV150526. 5. The principal metabolites in bile were identified as glucuronide conjugates of the carboxylic acid, whereas in rat urine the main metabolite was a sulphate conjugate of an aromatic oxidation metabolite. Multiple glucuronide peaks were observed and identified as isomeric glucuronides and their anomers arising from acyl migration and muta-rotation.     

Pharmacokinetics,metabolism and excretion of the glycine antagonist GV150526A in rat and dog

1. The pharmacokinetics, metabolic fate and excretion of 3-[-2(phenylcarbamoyl) ethenyl-4,6-dichloroindole-2-carboxylic acid (GV150526), a novel glycine antagonist for stroke, in rat and dog following intravenous administration of [C14]-GV150526A were investigated. 2. Studies were also performed in bile duct-cannulated animals to confirm the route of elimination and to obtain more information on metabolite identity. 3. Metabolites in plasma, urine and bile were identified by HPLC-MS/MS and NMR spectroscopy. 4. GV150526A was predominantly excreted in the faeces via the bile, with only trace metabolites of radioactivity in urine (< 5%). Radioactivity in rat bile was predominantly due to metabolites, whereas approximately 50% of the radioactivity in dog bile was due to parent GV150526. 5. The principal metabolites in bile were identified as glucuronide conjugates of the carboxylic acid, whereas in rat urine the main metabolite was a sulphate conjugate of an aromatic oxidation metabolite. Multiple glucuronide peaks were observed and identified as isomeric glucuronides and their anomers arising from acyl migration and muta-rotation.     

Alternate strategies to obtain mass balance without the use of radiolabeled compounds: application of quantitative fluorine (19F) nuclear magnetic resonance (NMR) spectroscopy in metabolism studies

Nuclear magnetic resonance (NMR) spectroscopy is playing an increasingly important role in the quantitation of small and large molecules. Recently, we demonstrated that (1)H NMR could be used to quantitate drug metabolites isolated in submilligram quantities from biological sources. It was shown that these metabolites, once quantitated by NMR, were suitable to be used as reference standards in quantitative LC/MS-based assays, hence circumventing the need for radiolabeled material or synthetic standards to obtain plasma exposure estimates in humans and preclinical species. The quantitative capabilities of high-field NMR is further demonstrated in the current study by obtaining the mass balance of fluorinated compounds using (19)F-NMR. Two fluorinated compounds which were radio-labeled with carbon-14 on metabolically stable positions were dosed in rats and urine and feces collected. The mass balance of the compounds was obtained initially by counting the radioactivity present in each sample. Subsequently, the same sets of samples were analyzed by (19)F-NMR, and the concentrations determined by this method were compared with data obtained using radioactivity counting. It was shown that the two methods produced comparable values. To demonstrate the value of this analytical technique in drug discovery, a fluorinated compound was dosed intravenously in dogs and feces and urine collected. Initial profiling of samples showed that this compound was excreted mainly unchanged in feces, and hence, an estimate of mass balance was obtained using (19)F-NMR. The data obtained by this method was confirmed by additional quantitative studies using mass spectrometry. Hence cross-validations of the quantitative (19)F-NMR method by radioactivity counting and mass spectrometric analysis were demonstrated in this study. A strategy outlining the use of fluorinated compounds in conjunction with (19)F-NMR to understand their routes of excretion or mass balance in animals is proposed. These studies demonstrate that quantitative (19)F-NMR could be used as an alternate technique to obtain an estimate of the mass balance of fluorinated compounds, especially in early drug development where attrition of the compounds is high, and cost savings could be realized through the use of such a technique rather than employing radioactive compounds. The potential application of qNMR in conducting early human ADME studies with fluorinated compounds is also discussed.     

Urinary metabolites of a novel quinoxaline non-nucleoside reverse transcriptase inhibitor in rabbit, mouse and human: identification of fluorine NIH shift metabolites using NMR and tandem MS

1. The urinary metabolites of (S)-2-ethyl-7-fluoro-3-oxo-3,4-dihydro-2H-quinoxaline-carboxylic acid isopropylester (GW420867X) have been investigated in samples obtained following oral administration to rabbit, mouse and human. GW420867X underwent extensive biotransformation to form hydroxylated metabolites and glucuronide conjugates on the aromatic ring, and on the ethyl and isopropyl side-chains in all species. In rabbit urine, a minor metabolite was detected and characterized as a cysteine adduct that was not observed in mouse or man. 2. The hydroxylated metabolites and corresponding glucuronide conjugates were isolated by semi-preparative HPLC and characterized using NMR, LC-NMR and LC-MS/MS. The relative proportions of fluorine-containing metabolites were determined in animal species by 19F-NMR signal integration. 3. The fluorine atom of the aromatic ring underwent NIH shift rearrangement in the metabolites isolated and characterized in rabbit, mouse and human urine. 4. The characterization of the NIH shift metabolites in urine enabled the detection and confirmation of the presence of these metabolites in human plasma.     

Quantitative studies on the urinary metabolic fate of 2-chloro-4-trifluoromethylaniline in the rat using 19F-NMR spectroscopy and directly coupled HPLC-NMR-MS

1. The metabolism and urinary excretion of 2-chloro-4-trifluoromethylaniline has been studied in the rat using 19F-NMR spectroscopy and directly coupled HPLC-NMR-MS methods. The compound was dosed to three male Sprague-Dawley rats (50 mg kg(-1) i.p.) and urine collected over 0-8, 8-24 and 24-48 h post-dosing. 2. A total urinary recovery of 56.3+/-2.2% of the dose was achieved up to 48 h after dosing. The major metabolite in the urine was identified as 2-amino-3-chloro-5-trifluoromethylphenylsulphate accounting for a total of 33.5+/-2.2% of the dose. 3. Further metabolites detected and characterized included 2-chloro-4-trifluoromethylphenylhydroxylamine glucuronide (13.2+/-0.5% of the dose), 2-amino-3-chloro-5-trifluoromethylphenylglucuronide (3.8+/-0.4% of the dose) and 2-chloro-4-trifluoromethylaniline-N-glucuronide (3.6+/-0.1% of the dose). Several minor metabolites were also found and identified, including 2-chloro-4-trifluoromethylphenylsulphamate, which together accounted for 2.1+/-0.4% of the dose. 4. Directly coupled HPLC-NMR-MS and 19F-NMR spectroscopy is shown to provide an efficient approach for the unequivocal and rapid determination of the quantitative urinary metabolic fate and excretion balance of a fluorinated xenobiotic without the necessity for specific radiolabelling.     

Urinary metabolites of a novel quinoxaline non-nucleoside reverse transcriptase inhibitor in rabbit,mouse and human: identification of fluorine NIH shift metabolites using NMR and tandem MS

1. The urinary metabolites of (S)-2-ethyl-7-fluoro-3-oxo-3,4-dihydro-2H-quinoxaline-carboxylic acid isopropylester (GW420867X) have been investigated in samples obtained following oral administration to rabbit, mouse and human. GW420867X underwent extensive biotransformation to form hydroxylated metabolites and glucuronide conjugates on the aromatic ring, and on the ethyl and isopropyl side-chains in all species. In rabbit urine, a minor metabolite was detected and characterized as a cysteine adduct that was not observed in mouse or man. 2. The hydroxylated metabolites and corresponding glucuronide conjugates were isolated by semi-preparative HPLC and characterized using NMR, LC-NMR and LCMS/MS. The relative proportions of fluorine-containing metabolites were determined in animal species by ¹⁹F-NMR signal integration. 3. The fluorine atom of the aromatic ring underwent NIH shift rearrangement in the metabolites isolated and characterized in rabbit, mouse and human urine. 4. The characterization of the NIH shift metabolites in urine enabled the detection and confirmation of the presence of these metabolites in human plasma.     

Study on metabolism of pyridonecarboxylic acid II. Determination of metabolites of (+-)-7-(3-amino-1-pyrrolidinyl)-6-fluoro-1-(2,4-difluorophenyl)- 1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylic acid p-toluenesulfonate hydrate (T-3262) in blood, urine, bile, and feces

The fate of (+-)-7-(3-amino-1-pyrrolidinyl)-6-fluoro-1-(2,4-difluorophenyl-1,4- dihyro-4-oxo-1,8-naphthyridine-3-carboxylic acid p-toluenesulfonate hydrate (T-3262) was studied using T-3262 and 14C-T-3262 in various animals. 1. Metabolites in serum and urine were assayed for mouse, rat, rabbit, dog and monkey following oral administration of T-3262. In serum, besides unchanged T-3262 base, T-3262A (N-acetylated) was detected in rat, rabbit and monkey; T-3262B (deamino-hydroxylated) was detected in monkey. In urine, unchanged T-3262 base was excreted mainly. But a few of metabolites (T-3262A, T-3262B, T-3262 glucuronide, T-3262A glucuronide, T-3262B glucuronide, and unknown compound M-1) were detected, and species difference existed in types of metabolites. 2. Metabolites in bile and feces were assayed for mouse and rat following oral administration of T-3262 and 14C-T-3262. Metabolites in bile were similar to the urine, but the volume of T-3262A and T-3262A glucuronide was larger than in urine. In feces, the excreted compounds mainly consisted of unchanged T-3262 base. 3. p-Toluenesulfonic acid, which is the counter acid for T-3262 base, was absorbed following the oral administration of T-3262, and excreted in urine in the unchanged form.     

The metabolism of (-)-3-phenoxy-N-methylmorphinan in dogs

The disposition of radioactive (-)-3-phenoxy-N-methyl[2-3H]morphinan in dogs after oral administration has been investigated. Unchanged drug was not found in bile, urine, or feces. Excretion of total radioactivity in feces ranged from 67 to 78% of an oral dose. Two unconjugated metabolites were isolated from feces and identified by NMR and GC/MS. Both were substituted on the phenoxy group; they were found to be the p-hydroxy (pOH-PMM) and the m-methoxy-p-hydroxy (mOCH3-pOH-PMM) metabolites. Further, levorphanol and norlevorphanol were identified in feces both as free and conjugated metabolites, as well as a small amount of levomethorphan. Urine contained mostly unknown metabolites and conjugated levorphanol and pOH-PMM. Although the glucuronide of mOCH3-pOH-PMM was the major metabolite in bile, smaller amounts of the glucuronide and sulfate conjugated of both levorphanol and pOH-PMM were also found. Estimates for the total urinary and fecal excretion (as percentages of the dose) by two dogs for the five known metabolites were as follows: levorphanol, 18.8-21.5%; pOH-PMM, 14.4-20.6%; mOCH3-pOH-PMM, 14.9%; norlevorphanol, 2.8-6.1%; levomethorphan, 0.5%. Two of these metabolites, pOH-PMM and levorphanol, are potent analgesics.     

Biotransformation of nevirapine, a non-nucleoside HIV-1 reverse transcriptase inhibitor, in mice, rats, rabbits, dogs, monkeys, and chimpanzees.

The study objectives were to characterize the metabolism of nevirapine (NVP) in mouse, rat, rabbit, dog, monkey, and chimpanzee after oral administration of carbon-14-labeled or -unlabeled NVP. Liquid scintillation counting quantitated radioactivity and bile, plasma, urine, and feces were profiled by HPLC/UV diode array and radioactivity detection. Metabolite structures were confirmed by UV spectral and chromatographic retention time comparisons with synthetic metabolite standards, by beta-glucuronidase incubations, and in one case, by direct probe electron impact ionization/mass spectroscopy, chemical ionization/mass spectroscopy, and NMR. NVP was completely absorbed in both sexes of all species except male and female dogs. Parent compound accounted for <6% of total urinary radioactivity and <5.1% of total fecal radioactivity, except in dogs where 41 to 46% of the radioactivity was excreted as parent compound. The drug was extensively metabolized in both sexes of all animal species studied. Oxidation to hydroxylated metabolites occurred before glucuronide conjugation and excretion in urine and feces. Hydroxylated metabolites were 2-, 3-, 8-, and 12-hydroxynevirapine (2-, 3-, 8-, and 12-OHNVP). 4-carboxynevirapine, formed by secondary oxidation of 12-OHNVP, was a major urinary metabolite in all species except the female rat. Glucuronides of the hydroxylated metabolites were major or minor metabolites, depending on the species. Rat plasma profiles differed from urinary profiles with NVP and 12-OHNVP accounting for the majority of the total radioactivity. Dog plasma profiles, however, were similar to the urinary profiles with 12-OHNVP, its glucuronide conjugate, 4-carboxynevirapine, and 3-OHNVP glucuronide being the major metabolites. Overall, the same metabolites are formed in animals as are formed in humans.     

Use of 19F-nuclear magnetic resonance and gas chromatography-electron capture detection in the quantitative analysis of fluorine-containing metabolites in urine of sevoflurane-anaesthetized patients

1: The use of fluorine-19 nuclear magnetic resonance (19F-NMR) and gas chromatography-electron capture detection (GC-ECD) in the analysis of fluorine-containing products in the urine of sevoflurane-exposed patients was explored. 2: Ten patients were anaesthetized by sevoflurane for 135-660 min at a flow rate of 6 l min(-1). Urine samples were collected before, directly after and 24 h after discontinuation of anaesthesia. 3: 19F-NMR analysis of the urines showed the presence of several fluorine-containing metabolites. The main oxidative metabolite, hexafluoroisopropanol (HFIP)-glucuronide, showed two strong quartet signals in the 19F-NMR spectrum. HFIP concentrations after beta-glucuronidase treatment were quantified by (19)F-nuclear magnetic resonance. Concentrations directly after and 24 h after discontinuation of anaesthesia were 131 +/- 41 (mean +/- SEM) and 61 +/- 19 mol mg(-1) creatinine, respectively. Urinary HFIP excretions correlated with sevoflurane exposure. 4: Longer scanning times enabled the measurement of signals from two compound A-derived metabolites, i.e. compound A mercapturic acid I (CAMA-I) and compound A mercapturic acid II (CAMA-II), as well as products from beta-lyase activation of the respective cysteine conjugates of compound A. The signals of the mercapturic acids, 3,3,3-trifluoro-2-(fluoromethoxy)-propanoic acid and 3,3,3-trifluorolactic acid were visible after combining and concentrating the patient urines. CAMA-I and -II excretions in patients were completed after 24 h. 5: Since 19F-nuclear magnetic resonance is not sensitive enough, urinary mercapturic acids concentrations were quantified by gas chromatography-electron capture detection. CAMA-I and -II urinary concentrations were 2.3 +/- 0.7 and 1.4 +/- 0.4 mol mg(-1) creatinine, respectively. Urinary excretion of CAMA-I showed a correlation with sevoflurane exposure, whereas CAMA-II did not. 6. The results show that 19F-nuclear magnetic resonance is a very selective and convenient technique to detect and quantify HFIP in non-concentrated human urine. 19F-nuclear magnetic resonance can also be used to monitor the oxidative biotransformation of sevoflurane in anaesthetized patients. Compound A-derived mercapturic acids and 3,3,3-trifluoro-2-(fluoromethoxy)-propanoic acid and 3,3,3-trifluorolactic acid, however, require more sensitive techniques such as gas chromatography-electron capture detection and/or gas chromatography-mass spectrometry for quantification.